Stereoselective synthesis of nucleoside analogues

ABSTRACT

The invention is a process for stereoselectively producing a dioxolane nucleoside analogue from an anomeric mixture of beta and alpha anomers represented by the following formula A or formula B:wherein R is selected from the group consisting of C1-6 alkyl and C6-15 aryl and Bz is benzoyl. The process comprises hydrolyzing said mixture with an enzyme selected from the group consisting of Protease N, Alcalase, Savinase, ChiroCLEC-BL, PS-30, and ChiroCLEC-PC to stereoselectively hydrolyze predominantly one anomer to form a product wherein R1 is replaced with H. The process also includes the step of separating the product from unhydrolyzed starting material. Additionally, the functional group at the C4 position is stereoselectively replaced with a purinyl or pyrimidinyl or derivative thereof.

This application is a continuation of Ser. No. 09/779,853, filed Feb. 9,2001, now U.S. Pat. No. 6,541,625.

This application claims the benefit of U.S. Provisional Application No.60/181,977, filed Feb. 11, 2000, the entire disclosure of which ishereby incorporated by reference.

FIELD OF THE INVENTION

The present invention relates generally to a novel method for thepreparation of nucleoside analogues and their precursors and moreparticularly to a method of preparing a nucleoside analogue by the useof specific enzymes to stereoselectively produce dioxolane nucleosideanalogues or their precursors.

BACKGROUND OF THE INVENTION

An important class of pharmacological agents relate to3′-oxa-substituted 2′,3′-dideoxynucleoside analogues (“dioxolanenucleoside analogues”). These compounds include9-(β-D-2-hydroxymethyl-1,3-dioxolan-4-yl)-2-aminopurine (β-D-DAPD);9-(β-D-2-hydroxymethyl-1,3-dioxolan-4-yl)-guanine (β-D-DXG);1-(β-L-2-hydroxymethyl-1,3-dioxolan-4-yl)-thymine (Dioxolane-T); and1-(β-L-2-hydroxymethyl-1,3-dioxolan-4-yl)-cytidine (β-L-OddC) which haveknown antiviral and antitumor activity.

As shown in the following dioxolane structure, dioxolanes have twochiral centers corresponding to the substituted carbons 2 and 4 of thedioxolane ring (C2 and C4 respectively). Thus each compound can exist asfour different stereoisomers depending on the position of bothsubstituents with respect to the dioxolane ring.

The stersoisomers of a dioxolane nucleoside analogue are represented bythe following diagrams where the letter B represents a purine orpyrimidine base or an analogue or derivative of a purine or pyrimidinebase as defined herewith.

For the purpose of consistency, the same stereochemical designation willbe used even when the hydroxymethyl moiety or the base moiety (B) isreplaced with another substituent group.

Chiral synthetic methods have improved over the past several years withrespect to synthetic techniques that result in single stereoisomercompounds. However, there is a present need to find novel syntheticmethods which can be widely used to form a particular stereoisomer withgreater efficiency and purity.

For example, for many years a person of ordinary skill in the art coulduse enzymes to separate enantiomers of dioxolane compounds. However,there is still a need in the art to produce a dioxolane nucleosideanalogue using a step of separating an anomeric mixture of certaindioxclane precursors to produce an end product with greater efficiencyand purity.

Because stereochemically pure dioxolane nucleosides are an importantclass of compounds due to their known antiviral activity and anticanceractivity, there is a need for other inexpensive and efficientstereoselective methods for their preparation. The present inventionsatisfies this and other needs.

SUMMARY OF THE INVENTION

The present invention provides a novel process for making dioxolanenucleoside analogues with a high degree of steric purity, greaterefficiency and higher yields.

Specifically, the present invention provides a process for makingdioxolane nucleoside analogues with a high degree of steric purity whichincludes the use of certain hydrolytic enzymes for separating β and αanomers from an anomeric mixture represented by the following formula Aor formula B:

wherein R₁ is selected from the group consisting of C₁₋₆ alkyl and C₆₋₁₅aryl; Bz is Benzoyl.

The process involves the step of hydrolyzing the mixture of compoundsrepresented by formula A and/or formula B with an enzyme selected fromthe group consisting of Protease N (Bacillus subtilis protease),Alcalase® (Subtilisin Carlsberg protease), Savinase® (Bacillus lentussubtilisin protease), ChiroCLEC™-BL (Bacillus licheniformis Subtilisinprotease), PS-30 (Pseudomonas cepacia lipase), and ChiroCLEC™-PC(Pseudomonas cepacia lipase). The process stereoselectively hydrolysespredominantly one anomer to form a product where R₁ of formula A andformula B is replaced with H. The other anomer remains substantiallyunhydrolysed. The process also comprises separating the hydrolyzedproduct from unhydrolysed starting material.

According to one embodiment of the invention, the aforementioned stepsof hydrolyzing and separating results in an isolated starting materialhaving an anomeric purity of at least 97% β-anomer. In an additionalembodiment, the aforementioned steps of hydrolyzing and separatingresults in an isolated starting material having an anomeric purity of atleast 98% β-anomer. In an additional embodiment, the aforementionedsteps of hydrolyzing and separating results in an isolated startingmaterial having an anomeric purity of at least 98.5% β-anomer. In anadditional embodiment, the aforementioned steps of hydrolyzing andseparating results in an isolated starting material having an anomericpurity of at least 98.8% β-anomer.

According to one embodiment of the invention, the aforementioned stepsof hydrolyzing and separating results in an isolated product having ananomeric purity of at least 97% α-anomer. In an additional embodiment,the aforementioned steps of hydrolyzing and separating results in anisolated product having an anomeric purity of at least 98% α-anomer. Inan additional embodiment, the aforementioned steps of hydrolyzing andseparating results in an isolated product having an anomeric purity ofat least 98.5% α-anomer. In an additional embodiment, the aforementionedsteps of hydrolyzing and separating results in an isolated producthaving an anomeric purity of at least 98.8% α-anomer.

In one embodiment, the β-anomer is the predominant product. In anotherembodiment, the α-anomer is the predominant product. In yet anotherembodiment, the β-L-enantiomer is the predominant product. In anadditional embodiment, the β-D-enantiomer is the predominant product. Inyet another embodiment, the α-L-enantiomer is the predominant product.In an additional embodiment, the α-D-enantiomer is the predominantproduct.

In one embodiment, the invention is a process for stereoselectivelypreparing a dioxolane nucleoside analogue by separating β and α-nomersfrom an anomeric mixture represented by formula A or formula B accordingto one of the above embodiments. The process further includes the stepof stereoselectively replacing the functional group at the C4 position(COOR₁) with a purinyl or pyrimidinyl or analogue or derivative selectedfrom the group consisting of:

In this embodiment, R₂, R₉ and R₁₁ are independently selected from thegroup consisting of hydrogen, C₁₋₆ alkyl, C₁₋₆ acyl and R₈C(O) whereinR₈ is hydrogen or C₁₋₆ alkyl. Additionally, R₃, R₄ and R₁₀ are eachindependently selected from the group consisting of hydrogen, C₁₋₆alkyl, bromine, chlorine, fluorine, iodine and CF₃; and R₅, R₆ and R₇are each independently selected from the group consisting of hydrogen,bromine, chlorine, fluorine, iodine, amino, hydroxyl and C₃₋₆cycloalkylamino. The process results in the production of astereochemical isomer of the dioxolane nucleoside analogue.

According to one embodiment, the process further includes the step ofstereoselectively replacing the functional group at the C4 position(COOR₁) with a purinyl or pyrimidinyl or derivative selected from thegroup consisting of:

In this embodiment, R₂ is selected from the group consisting ofhydrogen, C₁₋₆ alkyl, C₁₋₆ acyl and R₈C(O) wherein R₈ is hydrogen orC₁₋₆ alkyl. Additionally, R₃ and R₄ are each independently selected fromthe group consisting of hydrogen, C₁₋₆ alkyl, bromine, chlorine,fluorine, iodine and CF₃; and R₅, R₆ and R₇ are each independentlyselected from the group consisting of hydrogen, bromine, chlorine,fluorine, iodine, amino, hydroxyl and C₃₋₆ cycloalkylamino. The processresults in the production of a stereochemical isomer of a dioxolanenucleoside analogue.

In another embodiment, the process further includes the step ofstereoselectively replacing the functional group at the C4 position(COOR₁) with a pyrimidinyl or analogue or derivative selected from thegroup consisting of:

In this embodiment, R₉ and R₁₁ are independently selected from the groupconsisting of hydrogen, C₁₋₆ alkyl, C₁₋₆ acyl and R₈C(O). Additionally,R₁₀ is selected from the group consisting of hydrogen, C₁₋₆ alkyl,bromine, chlorine, fluorine, iodine and CF₃. The process results in theproduction of a stereochemical isomer of a dioxolane nucleosideanalogue.

In another embodiment, the process comprises stereoselectively preparinga dioxolane nucleoside analogue by separating β and α anomers from ananomeric mixture represented by formula A or formula B according to oneof the above embodiments and further comprises stereoselectivelyreplacing the functional group at the C4 position (COOR₁) with a moietyselected from the group consisting of:

In another embodiment of the present invention, the process comprisesmaking a dioxolane nucleoside analogue by separating a compoundaccording to formula A or formula B. According to this embodiment, theprocess includes stereoselectively replacing the R group with a9-purinyl or 1-pyrimidinyl moiety or analogue or derivative thereof byacylating the second mixture to produce an acylated second mixture. Thisembodiment also includes the step of glycosylating the acetylated secondmixture with a purine or pyrimidine base or analogue or derivativethereof and a Lewis Acid to produce a dioxolane nucleoside analogue.

DETAILED DESCRIPTION OF THE INVENTION

The present invention involves a high yield process of separating β andα anomers from an anomeric mixture of dioxolane nucleoside analogueprecursors which provides higher yield and greater efficiency. In oneembodiment, this method is used in the production of dioxolanenucleoside analogues having a high degree of anomeric purity at lowercost. Additionally, another aspect of the present invention involvessynthesizing starting material having a higher degree of anomericpurity.

The present invention provides a process of preparing dioxolanenucleoside analogues having a predominant β-L-configuration usingenzymes, namely hydrolases. The procedure improves overall yield and hasrelatively few steps, thereby improving overall efficiency. The processinvolves the following steps.

A mixture of anomers represented by formula A or formula B is obtainedas described herein in Scheme 1.

In the above formula, R₁ is selected from the group consisting of H,C₁₋₆ alkyl and C₆₋₁₅ aryl and Bz is Benzoyl. The mixture is hydrolyzedwith an enzyme selected from the group consisting of Alcalase®(Subtilisin Carlsberg protease, Novo Nordisk), Savinase® (Bacilluslentus substilisin protease, Novo Nordisk), ChiroCLEC™-BL (Bacilluslicheniformis Subtilisin protease, Altus Biologics, Inc.), PS-30(Pseudomonas cepacia lipase, Amano), Protease N (Bacillus subtilisprotease, Amano) and ChiroCLEC™-PC (Pseudomonas cepacia lipase, AltusBiologics, Inc.). The hydrolyzing step stereoselectively hydrolyzes theα-anomer of the mixture of either formula A or formula B. The result isan unhydrolyzed β-anomer. The α-anomer can be separated easily from theβ-anomer. If an anomeric mixture of the compound of formula A isselected, the result is the production of the compound of formula C andformula D:

If an anomeric mixture of the compound of formula B is selected, theresult is the production of the compound of formula E and formula F:

The mixture (C)/(D) or (E)/(F) is then subjected to oxidativedecarboxylation which replaces the R₁ group with an acyl moiety. It isthen glycosylated with a purine or pyrimidine base or analogue orderivative thereof in the presence of a Lewis Acid. The final stepproduces a dioxolane nucleoside analogue in the β-L configuration forthe mixture (C)/(D) and a dioxolane nucleoside analogue in the β-Dconfiguration for the mixture (E)/(F).

At the outset, the following definitions have been provided asreference. Except as specifically stated otherwise, the definitionsbelow shall determine the meaning throughout the specification.

“Nucleoside” is defined as any compound which consists of a purine orpyrimidine base, linked to a pentose sugar.

“Dioxolane nucleoside analogue” is defined as any compound containing adioxolane ring as defined hereinafter linked to a purine or pyrimidinebase or analogue or derivative thereof. A “dioxolane ring” is anysubstituted or unsubstituted five member monocyclic ring that has anoxygen in the 1 and 3 positions of the ring as illustrated below:

“Purine or pyrimidine base” is defined as the naturally occurring purineor pyrimidine bases adenine, guanine, cytosine, thymine and uracil. Apurine or pyrimidine that is a moiety is a purinyl or pyrimidinyl,respectively.

“Alkyl” is defined as a substituted or unsubstituted, saturated orunsaturated, straight chain, branched chain or carbocyclic moiety,wherein the straight chain, branched chain or carbocyclic moiety can beoptionally interrupted by one or more heteroatoms (such as oxygen,nitrogen or sulfur). A substituted alkyl is substituted with a halogen(F, Cl, Br, I), hydroxyl, amino or C₆₋₂₀ aryl.

“Aryl” is defined as a carbocyclic moiety which can be optionallysubstituted or interrupted by one heteroatom (such as oxygen, nitrogenor sulfur) and containing at least one benzenoid-type ring (such asphenyl and naphthyl).

“Carbocyclic moiety” is defined as a substituted or unsubstituted,saturated or unsaturated, C₃₋₆ cycloalkyl wherein a substitutedcycloalkyl is substituted with a C₁₋₆ alkyl, halogen (i.e. F. Cl, Br,I), amino, carbonyl or NO₂.

A “derivative” of a purine or pyrimidine base refers to one of thefollowing structures:

wherein one or more of the pyrimidine H are substituted withsubstituents that are known in the art. In the above illustration, thebonds represented by a broken line are optional and are present only incases which require the bond to complete the valence of the ring atom.Substitutents bound to the ring members by a single bond include but arenot limited to halogen such as F, Cl, Br, I; an akyl such as lowerakyls; aryl; cyano carbamoyl; amino including primary, secondary andtertiary amino; and hydroxyl groups. Substituents bound to the carbonring atoms by a double bond include but are not limited to a ═O to forma carbonyl moiety in the ring. It is understood that when the ring isaromatic, some of the substitutions may form tautomers. The definitionshall include such tautomers.

“Analogue” of a purine or pyrimidine base refers to any derivative ofpurine or pyrimidine bases that is further modified by substituting oneor more carbon in the ring structure with a nitrogen.

“Stereoselective enzymes” are defined as enzymes which participate ascatalysts in reactions that selectively yield one specific stereoisomerover other stereoisomers.

“Anomeric purity” is defined as the amount of a particular anomer of acompound divided by the total amount of all anomers of that compoundpresent in the mixture multiplied by 100%.

“Alkoxy” is defined as an alkyl group, wherein the alkyl group iscovalently bonded to an adjacent element through an oxygen atom (such asmethoxy and ethoxy).

“Alkoxycarbonyl”, is defined as an alkoxy group attached to the adjacentgroup of a carbonyl.

“Acyl” is defined as a radical derived from a carboxylic acid,substituted (by a halogen, C₆₋₂₀ aryl or C₁₋₆ alkyl) or unsubstituted byreplacement of the —OH group. Like the acid to which it is related, anacyl radical may be aliphatic or aromatic, substituted (by halogen, C₁₋₆alkoxyalkyl, nitro or O₂) or unsubstituted, and whatever the structureof the rest of the molecule may be, the properties of the functionalgroup remain essentially the same (such as acetyl, propionyl,isobutanoyl, pivaloyl, hexanoyl, trifluoroacetyl, chloroacetyl andcyclohexanoyl).

“Alkoxyalkyl” is defined as an alkoxy group attached to the adjacentgroup by an alkyl group (such as methoxymethyl).

“Acyloxy” is defined as an acyl group attached to the adjacent group byan oxygen atom.

“Oxo” is defined as a ═O substituent bonded to a carbon atom.

“Hydroxy protecting group” is well known in the field of organicchemistry. Such protecting groups may be found in T. Greene, ProtectiveGroups in Organic Synthesis, (John Wiley & Sons, 1981). Examples ofhydroxy protecting groups include but are not limited to benzyl,benzoyl, substituted benzoyl, acetyl and substituted acetyl.

As noted above, one embodiment of the present invention is a process forseparating β and α anomers from an anomeric mixture represented by thefollowing formula A or formula B:

wherein R₁ is selected from the group consisting of C₁₋₆ alkyl and C₆₋₁₅aryl; Bz is Benzoyl.

Another embodiment of the present invention is a process for separatingβ and α anomers from an anomeric mixture represented by the followingformula A′ or formula B′:

wherein R₁ is selected from the group consisting of C₁₋₆ alkyl and C₆₋₁₅aryl; W is a hydroxy protecting group.

In one embodiment, the process stereoselectively hydrolysespredominantly the α-anomer to form a product where R₁ of formula A andformula B is replaced with H. The β-anomer remains substantiallyunhydrolyzed. The process also comprises separating the hydrolyzedproduct from unhydrolyzed starting material.

The process of making a β-L dioxolane nucleoside analogue begins withthe preparation of starting materials. Scheme 1 depicts the manufactureof a mixture that includes formula A or B.

A benzoyloxyacetaldehyde (formula 1A) is reacted with1,3-dioxolane-4-carboxylic acid-2,2-dimethyl-methyl ester (formula 1B)in approximately equimolar proportions. The dioxolane of formula 1B hasa chiral center at-the C4 carbon. The reaction occurs in a toluenesolvent. The mixture is heated to 58° C. The catalyst, PTSA, is added.The mixture is heated to a temperature between 64-67° C. A vacuum isapplied at 70 kPa, and the reaction proceeds for 40 minutes. Traces ofsolvent are then removed by high vacuum. The catalyst is removed byfiltration using a 1:1 ratio of Hexane:EtOAc as an eluent. In oneembodiment, the preferred filter is a silica gel pad. The resultingproduct is a crude oil containing a mixture of the compounds of formula1C and 1D wherein the ratio is 2:1 of (1C:1D), respectively.

It can be appreciated by a person of skill in the art that the reactionconditions can be adjusted to optimize the purity of the stereoisomers.In one embodiment of the present invention, the reaction of the compoundof formula 1A with the compound of formula 1B is done in the presence ofcatalyst in an amount between about 1.0 wt % and 10.0 wt % of thestarting material. In another embodiment the amount of catalyst isbetween about 2.5 wt % and about 5.5 wt % of the starting materials. Inyet another embodiment, the amount of catalyst is between about 3.0 wt %and about 5.0 wt %. In still another embodiment, the amount of catalystis between about 3.5%. and about 5.5%. In another embodiment, the amountof catalyst is between about 2.5 wt % and about 7.5 wt %. In anotherembodiment the amount of catalyst is about 5.0 wt %.

In an embodiment of the present invention, the reaction of the compoundof formula 1A with the compound of formula 1B is done at a temperatureranging from about 40° C. to about 80° C. In another embodiment of thepresent invention, the temperature ranges from about 50° C. to about 75°C. In still another embodiment, the temperature ranges from about 60° C.to about 70° C. In an additional embodiment, the temperature ranges fromabout 65° C. to about 79° C.

In an embodiment of the present invention, the reaction time between thecompound of formula 1A and the compound of formula 1B corresponds to aperiod ranging from about 30 minutes to about 2 hours. In yet anotherembodiment, the period ranges from about 30 minutes to about 1 hour. Instill another embodiment, the period ranges from about 30 minutes toabout 50 minutes.

It will be appreciated by a person of ordinary skill in the art that theC4 carbon is chiral. Because this carbon is not involved in thereaction, the chirality is preserved at that carbon. A starting materialcan be selected to have a (4S) or (4R) stereochemistry.

According to one embodiment, it is preferable that the resulting productis an anomeric mixture favoring the β-L configuration over the α-Lconfiguration. To achieve such a result, the starting materialrepresented by formula 1B (4S) is selected and shown below:

The reaction proceeds according to the principles described above. Theresulting product, according to one embodiment, will have an anomericpurity of the β-L anomer over the α-L anomer of greater than 55%,preferably 60% and more preferably 65%.

According to one embodiment, the present invention is a method ofseparating β-anomers from α-anomers according to the following Scheme 2:

According to one embodiment, a mixture of anomers is obtained asrepresented by formula 2A or formula 2B. A mixture represented byformula 2A or formula 2B can be obtained according to the reactiondescribed above or according to any method known in the art.

The reaction is prepared as follows: A portion of the materialcontaining a mixture of compounds represented by formula 2A and formula2B is weighed into a reaction vessel. According to one embodiment, about3.7% mmol of the mixture is added to 10 mL of 20% acetonitrile/aqueousbuffer. In another embodiment for a preparative scale reaction, about75.2 mmole of the mixture is added to about 200 ml of 20%acetonitrile/aqueous buffer. The buffer is a phosphate buffer with a pHbetween 7.0 and 7.5 and preferably 7.2. In another embodiment a 20%aqueous t-butyl methyl ether was used.

The enzyme is selected from the group consisting of Alcalase®(Subtilisin Carlsberg protease), Savinase® (Bacillus lentus subtilisinprotease), ChiroCLEC™-BL (Bacillus licheniformis Subtilisin protease),PS-30 (Pseudomonas cepacia lipase), Protease N (Bacillus subtilisprotease), and ChiroCLEC™-PC (Pseudomonas cepacia lipase). These enzymesare commercially available. Particularly, some of the materials can beobtained from the following sources: Savinase® and Alcalase® can beobtained from Novo Nordisk. ChiroCLEC™-BL and ChiroCLEC™ can be obtainedfrom Altus Biologics, Inc. PS-30 and Protease N can be obtained fromAmano Pharmaceutical.

The stereospecific enzyme selected is then added to begin the hydrolysisreaction. The enzymatic reaction hydrolyzes primarily the α-anomer byreplacing the R₁ group of the α-anomer of the compound of formula 2Bwith H to form the compound of formula 2C. The amount of the enzymeadded can be determined according to principles known by any person ofordinary skill in the art. According to another embodiment, about 500 mLwas added to begin the reaction. The rate and degree of hydrolysis wasmonitored by a pH-stat according to principles known in the art. As thecompound of formula 2B is hydrolyzed, the pH of the mixture decreases.Thus, the change in pH as monitored by a pH-stat corresponds to thecompleteness of the reaction.

If the reaction time is allowed to proceed longer than the optimalreaction time, the β-anomer may be converted resulting in lower chemicalyield of the final product. If the reaction time is too short, less thanoptimal amount of the α-anomer is converted resulting in a loweranomeric purity of the remaining unhydrolyzed reactant. According to oneembodiment, the reaction is allowed to proceed until 43% completion. Itwill be appreciated by a person of ordinary skill in the art that theexact degree of completion may change depending upon the reactant used,the enzyme used and other principles known to a person of ordinary skillin the art.

As noted, the ester starting material and the hydrolysed product areseparated by increasing the pH of the solution to more than pH 7.0 andin one embodiment below pH 7.5 with sodium bicarbonate and extractingwith ethyl acetate (for example, 3×80 mL). The unhydrolysed startingmaterial is extracted in the ethyl acetate and the hydrolysed productremains in salt form in the aqueous solution. The pH of the solution isthen adjusted to pH 2. The hydrolyzed product is further extracted withethyl acetate (for example, 3×80 mL). The reactants and the products aredried with MgSO₄, filtered and concentrated in-vacuo.

Additionally, the unhydrolysed product can be hydrolysed by proceduresknown in the art such as reaction with LiOH followed by acidification.

Because of the enzyme selectivity, the anomeric purity of the hydrolyzedand separated α-anomer is considerably high.

According to one embodiment of the invention, the aforementioned stepsof hydrolyzing and separating results in an isolated starting materialhaving an anomeric purity of at least 97% β-anomer. In an additionalembodiment, the aforementioned steps of hydrolyzing and separatingresults in an isolated starting material having an anomeric purity of atleast 98% β-anomer. In an additional embodiment, the aforementionedsteps of hydrolyzing and separating results in an isolated startingmaterial having an anomeric purity of at least 98.5% β-anomer. In anadditional embodiment, the aforementioned steps of hydrolyzing andseparating results in an isolated starting material having an anomericpurity of at least 98.8% β-anomer.

According to one embodiment of the invention, the aforementioned stepsof hydrolyzing and separating results in an isolated product having ananomeric purity of at least 97% α-anomer. In an additional embodiment,the aforementioned steps of hydrolyzing and separating results in anisolated product having an anomeric purity of at least 98% α-anomer. Inan additional embodiment, the aforementioned steps of hydrolyzing andseparating results in an isolated product having an anomeric purity ofat least 98.5% α-anomer. In an additional embodiment, the aforementionedsteps of hydrolyzing and separating results in an isolated producthaving an anomeric purity of at least 98.8% α-anomer.

In another embodiment, the procedure of Scheme 2 is followed except theanomeric mixture represented by formula 2A and 2B is replaced with ananomeric mixture represented by formula 2D and 2E, respectively.

According to this embodiment the α-anomer represented by formula 2E ishydrolyzed The result is the separation of the hydrolyzed α-anomerrepresented by formula 2F from the unhydrolyzed β-anomer represented byformula 2D.

In another embodiment, the procedure of Scheme 2 is followed except amixture represented by formula 2A and 2B is replaced with a mixture offour stereoisomers represented by formula 2G.

According to this embodiment, the α-anomer containing both D and Lenantiomers is hydrolyzed. The result is the separation of thehydrolyzed α-anomer containing both D and L enantiomers from theunhydrolyzed β-anomer containing both D and L enantiomers.

After hydrolysis, purification and oxidative decarboxylation, theresulting dioxolane ring can be linked with a purine or pyrimidine baseor analogue or derivative. There are several examples known by skilledartisan on how to link a purine or pyrimidine base or analogue orderivative to the dioxolane ring. For example, PCT Publ. No. WO/97/21706by Mansour et al. describes one method of stereoselectively attachingthe purine or pyrimidine base or analogue or derivative to a dioxolanering. WO/97/21706 is incorporated herein fully by reference.

According to the process disclosed in WO/97/21706 the starting materialis an acylated dioxolane ring. The starting material of the proceduredisclosed in WO/97/21706 can be obtained by oxidative decarboxylation ofa product of Scheme 2 discussed above. Oxidative decarboxylationdestroys the stereochemistry of the C4 carbon while preserving thestereochemistry of the C2 carbon.

As noted, the oxidative decarboxylation step occurs after the hydrolysisstep of Scheme 2. A compound having the desired stereochemistry on theC2 carbon is selected. For each mmol of compound that is processed, itis dissolved in between about 2.5 and about 4.0 mL of acetonitrile. Inanother embodiment, between about 3.0 and about 3.5 mL of acetonitrilewas added for each mmol of compound. In yet another embodiment, betweenabout 3.3 and about 3.4 mL of acetonitrile was added for each mmol ofcompound.

For each mmol of compound, between about 0.08 and about 0.12 mL ofpyridine was added. In another embodiment, between about 0.09 and about0.11 mL of pyridine was added for each mmol of compound. In yet anotherembodiment, approximately 0.1 mL of pyridine was added for each mmol ofcompound.

To this mixture, between 1.1 and 1.5 mmoles of Pb(OAc)₄ was added foreach mmol of compound. In another embodiment, between about 1.2 mmolesand about 1.4 mmoles of Pb(OAc)₄ is added for each mmol of compound. Inyet another embodiment, about 1.3 mmoles of Pb(OAc)₄ is added for eachmmol of compound.

Thereafter, the mixture was stirred for 18 hours at room temperature.Then, the mixture was poured into a saturated solution of NaHCO₃.Between approximately 2.0 and 3.0 mL of NaHCO₃ were used for each mmolof compound. In one embodiment, between about 2.5 mL and about 2.7 mL,and more preferably about 2.6 mL of NaHCO₃ was used for each mmol ofcompound. The solution was then stirred for an additional 30 minutes.The organic layer was separated from the aqueous layer by fourextractions of ethyl acetate. Extracts were combined, dried on anhydrousNa₂SO₄ and evaporated under a vacuum. Optionally, the crude can befurther purified by chromatography on silica gel using a gradient of0-15% ethyl acetate in hexane.

In one embodiment of the present invention, the oxidativedecarboxylation step is followed by glycosylation. The glycosylation isrepresented by the following Scheme 3.

The first step in the glycosylation procedure is to obtain a compoundwith the desired stereospecificity at the C2 carbon. According to oneembodiment, a compound having an S stereochemistry at the C2 carbon, asrepresented by the compound of formula 3A is preferred. The result isthat a higher ratio of the β-L anomer is in the product 3C. According toanother embodiment, a compound having an R stereochemistry at the C2carbon is preferred. The result is a product that has a higher ratio ofthe β-D anomer in the final product.

The compound of formula 3A is reacted with an iodosilane to produce thecompound of formula 3B. In one embodiment, the iodosilane isiodotrimethylsilane.

In another embodiment, the iodosilane is diiodosilane. Important to thereaction is that it occurs at low temperatures. According to oneembodiment, the temperature is preferably between 0° C. and −78° C.prior to glycosylation with silylated pyrimidine or purine base oranalogue or derivative thereof. According to another embodiment, thetemperature is between 0° C. and −14.9° C. prior to glycosylation withsilylated pyrimidine or purine base or analogue or derivative thereof.According to yet another embodiment, the temperature is between 0° C.and −78° C. prior to glycosylation with silylated purine base oranalogue or derivative selected from the group comprising:

wherein R₅, R₆ and R₇ are each independently selected from the groupconsisting of hydrogen, bromine, chlorine, fluorine, iodine, amino,hydroxyl and C₃₋₆ cycloalkylamino.

According to still another embodiment, the temperature is between 0° C.and −78° C. prior to glycosylation with silylated purine base oranalogue or derivative thereof selected from the group comprising:

The iodo intermediate represented by formula 3B is then dissolved indichloromethane and is cooled down to a temperature comparable to thetemperature of the reaction vessel.

A purine or pyrimidine base or analogue or derivative thereof is thenselected. According to one embodiment, the purine or pyrimidine base oranalogue or derivative thereof is selected from the following group:

wherein R₂, R₉ and R₁₁ are each independently selected from the groupconsisting of hydrogen, C₁₋₆ alkyl, C₁₋₆ acyl and R₈C(O) wherein R₈ ishydrogen or C₁₋₆ alkyl;

R₃, R₄ and R₁₀ are each independently selected from the group consistingof hydrogen, C₁₋₆ alkyl, bromine, chlorine, fluorine, iodine and CF₃;and

R₅, R₆ and R₇ are each independently selected from the group consistingof hydrogen, bromine, chlorine, fluorine, iodine, amino, hydroxyl andC₃₋₆ cycloalkylamino.

According to one embodiment, the purine or pyrimidine base or derivativeis selected from the group consisting of:

In this embodiment, R₂ is selected from the group consisting ofhydrogen, C₁₋₆ alkyl, C₁₋₆ acyl and R₈C(O) wherein R₈ is hydrogen orC₁₋₆ alkyl. Additionally, R₃ and R₄ are each independently selected fromthe group consisting of hydrogen, C₁₋₆ alkyl, bromine, chlorine,fluorine, iodine and CF₃; and R₅, R₆ and R₇ are each independentlyselected from the group consisting of hydrogen, bromine, chlorine,fluorine, iodine, amino, hydroxyl and C₃₋₆ cycloalkylamino.

In another embodiment, the purine or pyrimidine base or analogue orderivative thereof is selected from the group consisting of:

In this embodiment, R₉ and R₁₁ are independently selected from the groupconsisting of hydrogen, C₁₋₆ alkyl, C₁₋₆ acyl and, R₈C(O). Additionally,R₁₀ is selected from the group consisting of hydrogen, C₁₋₆ alkyl,bromine, chlorine, fluorine, iodine and CF₃.

The purine or pyrimidine or analogue or derivative thereof is persylatedby a sylating agent and ammonium sulphate followed by evaporation ofHMDS to form a persylated purine or pyrimidine base or analogue orderivative thereof herein referred to as the persylated base anddesignated as P in Scheme 3. According to one embodiment, the sylatingagent is selected from the group consisting of1,1,1,3,3,3-hexamethyldisilazane, trimethylsilyl triflate,t-butyldimethylsilyl triflate or trimethylsilyl chloride. In oneembodiment, the sylating agent is 1,1,1,3,3,3,-hexamethyldisilazane.

The persylated base P was dissolved in 30 mL of dichloromethane and wasadded to the iodo intermediate represented by formula 3B. The reactionmixture was maintained at between 0 and 78° C. for 1.5 hours then pouredonto aqueous sodium bicarbonate and extracted with dichloromethane (2×25mL). The organic phase was dried over sodium sulphate to obtain thecompound of formula 3C. As used in Scheme 3, the B represents a moietyof the purine or pyrimidine base or analogue or derivative thereof whichwas persylated in the above step to form P. The compound of formula 3Cwas removed by filtration and the solvent was evaporated in-vacuo toproduce a crude mixture. The product represented by formula 3C haspredominantly a 4S configuration at the C4 carbon with an anomericpurity of 80%. When the starting material is a compound represented byformula 3A, the product forms predominantly the β-L enantiomer having ananomeric purity of 80%.

Next, the compound of formula 3C is deprotected to produce the compoundof formula 3D. This can be accomplished by dissolving a compoundrepresented by formula 3C in methanol and then adding ammonia or sodiummethoxide. The deprotection step can also be done by other methods whichare well known by those skilled in the art. The product represented byformula 3D is purified by flash chromatography on silica-gel (5% MEOH inethylacetate). The deprotection step can also be done by other methodsthat are well known by a person skilled in the art.

In another embodiment, compounds of Scheme 1 may be prepared by analternative process which is shown below in Scheme 4.

Between about 1.0-1.4 eq of sulfuric acid was added in portions to alarge excess of water while stirred at a temperature between 0-5° C. Byway of example and not by limitation, if 9.06 mol of D-Serine represents1 equivalent of reactant, then between about 9.5-13.3 mol of sulfuricacid is added to 7.3 L of water. In another embodiment, between about1.1-1.3 eq of sulfuric acid was added to an excess of water. In afurther embodiment, 1.2 eq of sulfuric acid was added to an excess ofwater.

About 1 equivalent of D-Serine was added in one portion under vigorousstirring. Then, between about 1.0 and 1.4 eq. of aqueous sodium nitritewas added dropwise. The temperature was kept between 0-5° C. during theaddition time (about seven hours). The reaction vessel was stirredovernight at room temperature. The water was removed by vacuum and theresidue (D-glyceric acid) co-evaporated with toluene (3×1L). The residuewas then stirred with about 6L of an alcohol solvent for about 30minutes. According to one embodiment, the alcohol is of the formula R₁OHwherein R₁ is a C₁₋₄ alkyl. According to another embodiment, the alcoholis methanol or ethanol. The resulting solid was removed by filtration.The clear solution was stirred at room temperature for 30-40 hours, thealcohol removed by vacuum to yield a D-glycerate in the form of a yellowviscous syrup. The D-glycerate is then reacted with between about0.9-1.1 eq of a dialkyl acetal at a temperature of about 85-95° C.Examples of suitable dialkyl acetals include benzoyloxyacetaldehydedialkyl. Examples of suitable alkyls for the dialkyl acetal is methyland ethyl.

Then, between about 1 wt % and about 10 wt % of PTSA is added. Accordingto another embodiment, about 5 wt % PTSA is added. In anotherembodiment, about 0.02 eq. of solid PTSA is added. The reaction mixtureis kept under vacuum at a temperature between 85-95° C. for 2-3 hours.The mixture is then cooled to room temperature, diluted withethylacetate (250 mL) and poured onto saturated sodium bicarbonatesolution (250 mL) under stirring. The organic phase is separated and theaqueous phase concentrated, purified on a silica gel column eluting with5-10% ethylacetate/hexanes to yield the desired dioxolane as a lightyellow oil (about 59%) with β/α ratio of 2:1 or higher.

Alternatively, the reactants of step 3 of Scheme 4 can be substituted bycorresponding reactants of Scheme 1. For example, the D-glyceraterepresented by Formula 4C is replaced with an 1,3dioxolane-4-(4R)carboxylic acid-2,2-dimethyl alkyl ester represented byFormula 1B according to one embodiment. Additionally or alternatively,the dialkyl acetal represented by Formula 4D is replaced with abenzoyloxyaldehyde represented by Formula 1A. These substitutions do notrequire changing the reaction conditions substantially disclosed abovefor the third step of Scheme 4.

In a further embodiment of the present invention, the starting materialof Scheme 4 is L-Serine which produces an end product having anS-configuration at the C4 carbon of the dioxclane ring. Alternatively,the L-glycerate of Step 3 can be replaced with an 1,3dioxolane-4-(4S)carboxylic acid-2,2-dimethyl alkyl ester to produce anend product having predominantly an S-configuration at the C4 carbon ofthe resulting dioxolane ring.

EXAMPLE 1 Enzyme Catalyzed Hydrolytic Resolution of the Dioxolane MethylEster Using Savinase®

A 2:1 (β:α) anomeric mixture of (2-(S)-benzoyloxymethyl)-4-carboxylicacid-1,3-dioxolane methyl ester) (20 g, 75.2 mmol) was weighed into areaction vessel and was disolves with 40 mL of acetonitrile. 160 mL ofpH 7.2 phosphate buffer was added to form a suspension. Savinase® (5 mLwas added to begin the reaction and the rate and degree of hydrolysiswas monitored by HPLC analysis with ChiraCel OD column or a pH-statwhich maintained the pH at 7 by automatic titration with 1 M NaOH. Thereaction was terminated when the anomeric purity of the remaining esterreached 98% (about 8 hours). After the pH of the reaction mixture wasadjusted to pH 7.5 with 1 M NaOH, the remaining starting material esterwas extracted with ethyl acetate (3×80 mL). The aqueous phase wasadjusted to pH 6.0 and the product acid was extracted.

Both extracts were dried with MgSO₄, filtered and concentrated in-vacuo.By this method, we obtained the(2-(S)-benzoyloxymethyl)-4-(S)-carboxylic acid-1,3-diaxolanemethylester) with greater than 98% anomeric purity.

EXAMPLE 2 Purity of β-Anomer by NMR

Analysis was performed on a Varian Gemini 200 MHz NMR spectrometer inCDCl₃. The α-ester shows a triplet at 5.33 (³J=4.6 Hz) and the β-estershows a triplet upfield at 5.23 (³J=4.6 Hz). The α-acid shows a tripletat 5.33(³J=3.6 Hz), while the β-acid shows a broad singlet upfield at d5.19. We did not observe any epimerization of the substrate or productacid during work-up. By NMR analysis, the purity of the β-anomer isdetermined to have 98% anomeric purity.

EXAMPLE 3 Purity of the α-Anomer

The product acid is obtained from Example 1 after it was dried withMgSO₄, filtered and concentrated in-vacuo. It is analyzed for purity byNMR. The α-anomer is isolated with high anomeric purity.

EXAMPLE 4 Enzymatic Resolution of β-Anomer with Alcalase®

The procedures of Examples 1-2 were followed using Alcalase® as theenzyme to separate a 2:1 (β:α) anomeric mixture of(2-(S)-benzoyloxymethyl)-4-carboxylic acid-1,3-dioxolane methyl ester).The result is a β-anomer that has high anomeric purity.

EXAMPLE 5 Enzymatic Resolution of α-Anomer Alcalase®

The product acid is obtained from Example 4 after it is dried withMgSO₄, filtered and concentrated in-vacuo. The α-anomer is isolated withhigh anomeric purity.

EXAMPLE 6 Enzymatic Resolution of β-Anomer with ChiroCLEC™-BL

The procedures of Examples 1-2 were followed using ChiroCLEC™-BL as theenzyme to separate a 2:1 (β:α) anomeric mixture of(2-(S)-benzoyloxymethyl)-4-carboxylic acid-1,3-dioxalane methyl ester).The result is a β-anomer that has high anomeric purity.

EXAMPLE 7 Enzymatic Resolution of α-Anomer with ChiroCLEC™-BL

The product acid is obtained from Example 6 after it is dried withMgSO₄, filtered and concentrated in-vacuo. The α-anomer is isolated withhigh anomeric purity.

EXAMPLE 8 Enzymatic Resolution of β-Anomer with PS-30

The procedures of Examples 1-2 were followed using PS-30 as the enzymeto separate a 2:1 (β:α) anomeric mixture of(2-(S)-benzoyloxymethyl)-4-carboxylic acid-1,3-dioxolane methyl ester).The result is a β-anomer that has high anomeric purity.

EXAMPLE 9 Enzymatic Resolution of α-Anomer with PS-30

The product acid is obtained from Example 8 after it is dried withMgSO₄, filtered and concentrated in-vacuo. The α-anomer is isolated withhigh anomeric purity.

EXAMPLE 10 Enzymatic Resolution of β-Anomer with ChiroCLEC™-PC

The procedures of Examples 1-2 were followed using ChiroCLEC™-PC as theenzyme to separate a 2:1 (β:α) anomeric mixture of(2-(S)-benzoyloxymethyl)-4-carboxylic acid-1,3-dioxolane methyl ester).The result is a β-anomer that has high anomeric purity.

EXAMPLE 11 Enzymatic Resolution of α-Anomer with ChiroCLEC™-PC

The product acid is obtained from Example 10 after it is dried withMgSO₄, filtered and concentrated in-vacuo. The α-anomer is isolated withhigh anomeric purity.

EXAMPLE 12 Enzymatic Resolution of β-Anomer with Protease N

The procedures of Examples 1-2 were followed using Protease N as theenzyme to separate a 2:1 (β:α) anomeric mixture of(2-(S)-benzoyloxymethyl)-4-carboxylic acid-1,3-dioxolane methyl ester).The result is a β-anomer that has high anomeric purity.

EXAMPLE 13 Enzymatic Resolution of α-Anomer with Protease N

The product acid is obtained from Example 12 after it is dried withMgSO₄, filtered and concentrated in-vacuo. The α-anomer is isolated withhigh anomeric purity.

EXAMPLE 14 Preparation of2-(S)-Benzoyloxymethyl-4-(R)-iodo-1,3-dioxolane and2-(S)-Benzoyloxymethyl-4-(S)-iodo-1,3-dioxolane (Compound 14)

A mixture consisting of 2S-benzoyloxymethyl-4S acetoxy-1,3-dioxolane and2S-benzoyloxymethyl-4R-acetoxy-1,3-dioxolane in 1:2 ratio (6 g; 23.8mMol) was dried by azeotropic distillation with toluene in-vacuo. Afterremoval of toluene, the residual oil was dissolved in drydichloromethane (60 mL) and iodotrimethylsilane (3.55 mL; 1.05 eq.) wasadded at −78° C., under vigorous stirring. The dry-ice/acetone bath wasremoved after addition and the mixture was allowed to warm up to roomtemperature (15 min.). The product was2S-benzoyloxymethyl-4R-iodo-1,3-dioxolane and 2S-benzoyloxymethyl-4S-iodo-1,3-dioxolane.

It would be understood by a person of ordinary skill in the art that ifthe starting mixture was chosen consisting of 2-R-benzoyloxymethyl-4Sacetoxy-1,3-dioxolane and 2R-benzoyloxymethyl-4R-acetoxy-1,3-dioxolane.The resulting product is 2R -benzoyloxymethyl-4R-iodo-1,3-dioxolane and2R-benzoyloxymethyl-4S-iodo-1,3-dioxolane. Furthermore, the startingmaterial having a benzoyl substituent group instead of a benzyl wouldresult in a product having a benzoyl substituent and not a benzyl.

EXAMPLE 15 Synthesis of2-(S)-Benzoyloxymethyl-1,3-dioxolan-4-(S)-yl)-2-oxo-4-aminoacetyl-pyrimidine(Compound 15)

The previously prepared iodo intermediate (Compound 14) indichloromethane, was cooled down to −78° C. Persylated N-acetyl cytosine(1.1 eq) formed by reflux in 1,1,1,3,3,3-hexamethyl disilazane (HMDS)and ammonium sulphate followed by evaporation of HMDS was dissolved in30 mL of dichloromethane and was added to the iodo intermediate. Thereaction mixture was maintained at −78° C. for 1.5 hours then pouredonto aqueous sodium bicarbonate and extracted with dichloromethane (2×25mL). The organic phase was dried over sodium sulphate, the solid wasremoved by filtration and the solvent was evaporated in-vacuo to produce8.1 g of a crude mixture. β-L-4′-benzyl-2′-deoxy-3′-oxacytidine and itsα-L isomer were formed in a ratio of 5:1 respectively. This crudemixture was separated by chromatography on silica-gel (5% methanol inethylacetate) to generate the pure β-L (β) isomer (4.48 g).Alternatively, recrystallization of the mixture from ethanol produces4.92 g of pure β isomer and 3.18 g of a mixture of β and α-isomers in aratio of 1:1.

EXAMPLE 162-(S)-Benzoyloxymethyl-1,3-dioxolan-4-(S)-yl)-2-oxo-4-amino-pyrimidine(Compound 16)

The protected β-L isomer (4.4 g) (Compound 15) was suspended insaturated methanolic ammonia (250 mL) and stirred at room temperaturefor 18 hours in a closed vessel. The solvents were then removed in-vacuoto afford the deacetylated nucleoside in pure form.

EXAMPLE 172-(S)-hydroxymethyl-1,3-dioxolan-4-(S)-yl)-2-oxo-4-amino-pyrimidine(Compound 17)

β-L-4′-Benzyl-2′-deoxy-3′-oxacytidine (Compound 16) was dissolved inEtOH (200 mL) followed by addition of cyclohexene (6 mL) and palladiumoxide (0.8 g). The reaction mixture was refluxed for 7 hours then it wascooled and filtered to remove solids. The solvents were removed from thefiltrate by vacuum distillation. The crude product was purified by flashchromatography on silica-gel (5% MeOH in EtOAc) to yield a white solid(2.33 g; 86% overall yield). α_(D) ²²=−46.7° (c=0.285; MeOH)m.p.=192-194° C.

The following examples 18-20 illustrate a method of preparing thestarting material of example 1 (2-(S)-benzoyloxymethyl-4-carboxylicacid-1,3-dioxolane methyl ester).

EXAMPLE 18 Preparation of D-Glyceric Acid (Compound 18)

Portions of sulfuric acid (297 mL; 11.14 mol; 1.23 eq) was added to alarge excess of water (7,300 mL) under stirring and cooling (0-5° C.).D-Serine (952 g;9.06 mol;1 eq) was added in one portion under vigorousstirring, followed by dropwise addition of aqueous sodium nitrite (769g;11.14 mol; 1.23 eq in 3,060 mL water). Temperature was kept between0-5° C. during the addition time (seven hours). The reaction vessel wasstirred overnight at room temperature and the reaction monitored by TLC(ninhydrin). In order to complete the reaction, additional sulfuric acid(115 mL; 4.31 mol; 0.47 eq) and aqueous sodium nitrite (255 g; 3.69 mol;0.4 eq in 1,100 mL water) was added, keeping the reaction vesseltemperature between 0-5° C. The reaction vessel was then kept understirring at room temperature for another 18 hours. Nitrogen was bubbledthrough the solution for one hour and the water removed by vacuum,keeping the reaction vessel temperature between 28-30° C. The residue(D-glyceric acid) was co-evaporated with toluene (3×1L).

EXAMPLE 19 Preparation of D-Methyl Glycerate (Compound 19)

D-glyceric acid was stirred with methanol (6L) for 30 minutes and thesolid removed by filtration. The clear solution was stirred at roomtemperature for 35-38 hours and the reaction monitored by TLC (DCM/MeOH8:2 Rf=0.63). Methanol was removed by vacuum to yield a yellow viscoussyrup (1,100 g).

EXAMPLE 20 Preparation of2-(R,S)-benzoyloxymethyl-4-R-methylcarboxylate-1,3-dioxolane (Compound20)

A mixture of benzoyloxyacetaldehyde dimethyl acetal (146 g, 95%, 0.66mole, 1 eq) and D-methyl glycerate (99 g, 0.82 mole, 1.25 eq) was heatedto 90° C., followed by the addition of solid PTSA (2.75 g, 0.145 moles,0.022 eq). The reaction mixture was kept under vacuum (water aspirator)at 90-95° C. for 2.5 hours (TLC, Hexanes/Ethylacetate 1:1, Rf-0.47). Thereaction mixture was cooled down to room temperature, diluted withethylacetate (250 mL) and poured onto saturated NaHCO3 solution (250 mL)under stirring. The organic phase was separated and the aqueous phasewas extracted one with ethylacetate (150 mL). The combined organic phasewas concentrated and purified on a silica gel column eluting with 5-10%ethylacetate/hexanes to yield 112.4 g of the desired product as a lightyellow oil (59%) with β/α ratio of 2.1:1. The β-anomer of compound 20can be then separated from the α-anomer of compound 20 according toExamples 1-3, 4-5, 6-7, 8-9, 10-11, or 12-13.

EXAMPLE 21 Preparation ofβ2-(R)-benzoyloxymethyl-1,3-dioxolane-4-(R)-carboxylic acid (Compound21)

β2-(R)-benzoyloxymethyl-1,3-dioxolane-4-(R)-methylcarboxylate-1,3-dioxolane(15.327 g; 57.57 mmol) is dissolved in THF (60 mL) then water (15 mL)was added under stirring. The internal temperature was set to 20° C.Then, a solution of LiOH (2.41 g; 57.57 mmol) in water (15 mL) was addeddropwise over 7 minutes. The reaction mixture was stirred at 22° C. foran additional 40 minutes. THF was removed under vacuum, and the residuediluted with water (70 mL). The resulting solution was extracted withdichloromethane (2×35 mL). The aqueous phase was acidified by 30% H₂SO₄(9.5 mL) under tight pH-meter control (initial pH:8.36 to 3.02) thenextracted with DCM (4×60 mL). The organic phases were combined and thesolvent removed under vacuum to furnish a light green syrup (14.26 g)which was kept under vacuum overnight.

EXAMPLE 22 Preparation ofβ2-(R)-benzoyloxymethyl-4-(R,S)-methylcarboxylate-1,3-dioxolane(Compound 22)

Lead tetraacetate (944, 8 g; 2,024 mole; 1,2 eq) was added portion-wiseto an acetonitrile (6.8 L) solution of the acid (425,5 g; 1,687 mole;1,0 eq) and pyridine (193 mL) in an ice bath. The reaction vessel wasallowed to warm up to room temperature and stirred. The reaction waschecked by TLC (hexanes:ethyl acetate 6:4). It was filtered through asmall pad of celite (about 1 inch). Then, the filtrate was poured onto 5L of saturated aqueous sodium bicarbonate solution (reaction mixtureturned brown), and the pH as adjusted to 8 by adding solid sodiumbicarbonate. The filtrate was again filtered through a small pad ofcelite (about 1 inch) to remove the black lead salts to yield a paleyellow mixture. The organic phase was separated and the aqueous phasewas extracted with ethylacetate (4×2L). The combined organic phase wasconcentrated, and the oil obtained was co-evaporated with toluene (3×2L)to yield a brown syrup.

This syrup (374 g) was further purified by filtering through a small padof silica gel (1 g crude; 2 g silica), eluting with 3.5 L of the solventmixture (ethyl acetate:hexanes 8:2) to yield 332,3 g (74%) of pureproduct. This last filtration step is optional.

EXAMPLE 23 Preparation of9-(2-(R)-benzoyloxymethyl-1,3-dioxolan-4-yl)-6-chloro-2-amino purine(Compound 23a) and9-(2-(R)-benzoyloxymethyl-1,3-dioxolan-4-yl)-6-iodo-2-amino purine(Compound 23b)

TMSI (28.2 mL; 198.12 mol eq) was added dropwise to a dichloromethane(750 mL) solution of the sugar(2-(R)-benzoyloxymethyl-4-carboxyl-1,3-dioxolane)(52.75 g; 198.12 mmol;1 eq) at 15° C. After 2.5 hr at 15° C., silylated 2-amino-6-chloropurine(62 g; 198 mmol; 1 eq) was added to the reaction mixture as a solid. Thestirring was continued at the same temperature for another 2.5 hr. Thereaction mixture was allowed to warm up slowly to room temperaturefollowed by continued stirring for 40 hr at room temperature. Then, themixture was poured onto aq NaHCO₃ solution (1 L). It was stirred for 20min with Na₂S₂O₃ and filtered through a small pad celite. Then, theorganic phase was separated and the aqueous phase was extracted withdichloromethane (1×200 mL). The combined organic phases wereconcentrated to get 87 g of the crude. Column purification of the crudeon silica gel (450 g), eluting with ethylacetate/hexane (6:4) yielded67.7 g (81%; 1:1 chloro/iodo mixture) of the coupled product with β/αratio 2.3:1.

Alternatively, if the desired final product is the same compound butwith opposite stereochemistry (i.e. a 2:1 mixture of β:α stereoisomersin the L-configuration). The procedure discussed above is followed.However, the sugar 2-(R)-benzoyloxymethyl-4-carboxyl-1,3-dioxolane isreplaced with 2-(S)-benzoyloxymethyl-4-carboxyl-1,3-dioxolane.

EXAMPLE 24 Preparation of9-(2-(R)-benzoyloxymethyl-1,3-dioxolan-4-yl)-6-(N-cyclopropyl)amino-2-aminopurine (Compound 24)

A solution of the starting material (Compound 23: 6.3 g; 14.95 mmol; 1eq; average F.W.=421.52; Cl:I/1:1) in ethanol (100 mL) was refluxed at75-80° C. with cyclopropylamine (3.1 mL; 44.84 mmol; 3 eq) for 20 hrsand cooled to room temperature. The reaction mixture was concentrated,dissolved in dichloromethane (25 mL) and poured onto saturated aqueoussodium bicarbonate solution. After 10 min. of stirring, the organicphase was separated, and the aqueous phase was extracted withdichloromethane (2×15 mL). Then, the combined organic phase wasconcentrated to get a quantitative yield of the crude, which was thenpurified by column chromatography (silica gel, ethylacetate:MeOH98.5:2.5 and 95:5) to yield 5.3 g (89%) of the product as a β/α mixture.

EXAMPLE 25 Preparation of9-(2-(R)-hydroxymethyl-1,3-dioxolan-4-yl)-6-(N-cyclopropyl)amino-2-aminopurine (Compound 25)

The starting material (Compound 24: 3.3 g) was stirred with ammonia inMeOH (80 mL; 2M) for 20 hrs. Nitrogen was bubbled through the reactionmixture to remove the excess ammonia. Then, the solution wasconcentrated to yield the crude as a β/α mixture (β/α=2.3:1). The β/αisomers were separated by chromatography on silica gel using DCM/MeOH aseluent to yield 1.18 g (70% β isomer).

EXAMPLE 26 Preparation of9-(2-(R)-hydroxymethyl-1,3-dioxolan-4-yl-6-(N-2-cyclopropyl-2-aminomethoxyl)-2-aminopurine (Compound 26)

A solution of(2R)-2-benzoyloxymethyl-4-(2′-amino-6′-cyclopropylamino-purine-9′-yl)-1,3-dioxolane(480 mg) in 30 ml of saturated methanolic ammonia was stirred at roomtemperature for 18 h. The mixture was evaporated to dryness in vacuo.The residue was dissolved in 20 ml of water, washed twice with 10 ml ofmethylene chloride and lyophilized to give 283 mg of white solid in 80%yield. The resulting product had a mixture of β:α anomers having a ratioof about 2:1.

Alternatively, if the desired final product is the same compound butwith opposite stereochemistry (i.e. a 2:1 mixture of β:α stereoisomersin the L-configuration). The procedure discussed above is followed.However, when following the steps of Example 23, the sugar2-(R)-benzoyloxymethyl-4-carboxyl-1,3-dioxolane is replaced with2-(S)-benzoyloxymethyl-4-carboxyl-1,3-dioxolane.

EXAMPLE 27 Preparation of9-(2-(S)-hydroxymethyl-1,3-dioxolan-4-yl)-2-amino purine (Compound 27)

The procedure of Example 23 was performed. Thereafter 6.3 g of Compound23 was subject to hydrogenation conditions under 50 psi of hydrogen over10% Pd/c in 300 mL of ethanol containing 100 mL of triethylamine. After3 hours of shaking, the catalyst was removed by filtration. Then thesolvent was evaporated to yield a solid which was recrystallised to fromethanol-ether to give about 4 g of Compound 27 having about a 2:1mixture of β:α stereoisomers in the L-configuration.

Alternatively, if the desired final product is the same compound butwith opposite stereochemistry (i.e. about a 2:1 mixture of β:αstereoisomers in the D-configuration). The procedure discussed above isfollowed. However, when following the steps of Example 23, the sugar2-(S)-benzoyloxymethyl-4-carboxyl-1,3-dioxolane is replaced with2-(R)-benzoyloxymethyl-4-carboxyl-1,3-dioxolane.

EXAMPLE 28 Preparation of 9-(2-(S)hydroxymethyl-1,3-dioxolan-4-yl)-6-amino purine (Compound 28)

The procedures set forth in Examples 23 and 24 were performed. Howeverwhen following the steps of Example 23, the 1 equivalent of the silated2-amino-6-chloropurine is replaced with 1 equivalent of silated6-aminopurine. The result is a yield of9-(2-(S)-hydroxymethyl-1,3-dioxolan-4-yl)-6-amino purine having a β:αratio of about 2:1.

Alternatively, if the desired final product is the same compound butwith opposite stereochemistry (i.e. a 2:1 mixture of β:α stereoisomersin the D-configuration). The procedure discussed above is followed.However, when following the steps of Example 23, the sugar2-(S)-benzoyloxymethyl-4-carboxyl-1,3-dioxolane is replaced with2-(R)-benzoyloxymethyl-4-carboxyl-1,3-dioxolane.

EXAMPLE 29 Preparation of 9-(2-(S)hydroxymethyl-1,3-dioxolan-4-yl)-6,2-diamino purine (Compound 29)

The procedure of Example 23 was performed. Thereafter, 6 g of Compound23 was dissolved in 0.9 L of methanol saturated at 0° C. with dryammonia and the solution is heated in a steel bomb to 105° C. to 110° C.for 16 hours. The solution was evaporated to dryness and the residuepurified by chromatography on silica gel using chloroform-methanol (4:1)as the eluent to give about 3 g of crude Compound 29. The product can berecrystallised from methanol-ether to yield purified Compound 29 havinga β:α ratio of about 2:1.

Alternatively, if the desired final product is the same compound butwith opposite stereochemistry (i.e. a 2:1 mixture of β:α stereoisomersin the D-configuration). The procedure discussed above is followed.However, when following the steps of Example 23, the sugar2-(S)-benzoyloxymethyl-4-carboxyl-1,3-dioxolane is replaced with2-(R)-benzoyloxymethyl-4-carboxyl-1,3-dioxolane.

EXAMPLE 30 Preparation of 9-(2-(S)hydroxymethyl-1,3-dioxolan-4-yl)-6-oxo-2-amino purine (Compound 30)

The procedure of Example 23 was performed. Thereafter, about 6 g ofCompound 23 was dissolved in a mixture of 200 mL of methanol, 50 mL ofwater and 10 g of NaoH. The solution was heated under reflux for 5 hoursafter which time it was diluted with 300 mL of water and excesspyridinium sulfonate resin. The slurry was filtered, the resin washedwith water and the combined aqueous filtrates were evaporated to drynessin vacuo to leave a residue which was taken up in 50% aqueous methanol.The solution was treated with activated charcoal, filtered and thefiltrate evaporated to dryness in vacuo to give a solic residue that wasrecrystallized from ethanol water to yield pure compound 30 having a β:αratio of about 2:1.

Alternatively, if the desired final product is the same compound butwith opposite stereochemistry (i.e. a 2:1 mixture of β:α stereoisomersin the D-configuration). The procedure discussed above is followed.However, when following the steps of Example 23, the sugar2-(S)-benzoyloxymethyl-4-carboxyl-1,3-dioxolane is replaced with2-(R)-benzoyloxymethyl-4-carboxyl-1,3-dioxolane.

EXAMPLE 31 Preparation of 9-(2-(S)hydroxymethyl-1,3-dioxolan-4-yl)-2-oxo-4-amino-5-methyl pyrimidine(Compound 31)

The procedure of Example 23 was performed followed by the procedure ofExample 25. However, when following the steps of Example 23, the 1equivalent of the silated 2-amino-6-chloropurine is replaced with 1equivalent of silated 2-oxo-4-amino-5-methyl-pyrimidine. The result is ayield of9-(2-(S)-hydroxymethyl-1,3-dioxolan-4-yl)-2-oxo-4-amino-5-methylpyrimidine having a β:α ratio of about 2:1.

Alternatively, if the desired final product is the same compound butwith opposite stereochemistry (i.e. a 2:1 mixture of β:α stereoisomersin the D-configuration). The procedure discussed above is followed.However, when following the steps of Example 23, the sugar2-(S)-benzoyloxymethyl-4-carboxyl-1,3-dioxolane is replaced with2-(R)-benzoyloxymethyl-4-carboxyl-1,3-dioxolane.

EXAMPLE 32 Preparation of 9-(2-(S)hydroxymethyl-1,3-dioxolan-4-yl)-2-oxo-4-amino-5-fluoro pyrimidine(Compound 32)

The procedure of Example 23 was performed followed by the procedure ofExample 25. However, when following the steps of Example 23, the 1equivalent of the silated 2-amino-6-chloropurine is replaced with 1equivalent of silated 2-oxo-4-amino-5-fluoro-pyrimidine. The result is ayield of 9-(2-(S)hydroxymethyl-1,3-dioxolan-4-yl)-2-oxo-4-amino-5-fluoro pyrimidinehaving a β:α ratio of about 2:1.

Alternatively, if the desired final product is the same compound butwith opposite stereochemistry (i.e. a 2:1 mixture of β:α stereoisomersin the D-configuration). The procedure discussed above is followed.However, when following the steps of Example 23, the sugar2-(S)-benzoyloxymethyl-4-carboxyl-1,3-dioxolane is replaced with2-(R)-benzoyloxymethyl-4-carboxyl-1,3-dioxolane.

EXAMPLE 33 Preparation of 9-(2-(S)hydroxymethyl-1,3-dioxolan-4-yl)-2,4-dioxo pyrimidine (Compound 33)

The procedure of Example 23 was performed followed by the procedure ofExample 25. However, when following the steps of Example 23, the 1equivalent of the silated 2-amino-6-chloropurine is replaced with 1equivalent of silated 2,4-dioxo pyrimidine. The result is a yield of9-(2-(S)-hydroxymethyl-1,3-dioxolan-4-yl)-2,4-dioxo pyrimidine having aβ:α ratio of about 2:1.

Alternatively, if the desired final product is the same compound butwith opposite stereochemistry (i.e. a 2:1 mixture of β:α stereoisomersin the D-configuration. The above formula is followed. However, whenfollowing the steps of Example 23, the sugar2-(S)-benzoyloxymethyl-4-carboxyl-1,3-dioxolane is replaced with2-(R)-benzoyloxymethyl-4-carboxyl-1,3-dioxolane.

EXAMPLE 34 Preparation of 9-(2-(S)hydroxymethyl-1,3-dioxolan-4-yl)-2,4-dioxo-5-methyl pyrimidine (Compound34)

The procedure of Example 23 was performed followed by the procedure ofExample 25. However, when following the steps of Example 23, the 1equivalent of the silated 2-amino-6-chloropurine is replaced with 1equivalent of silated 2,4-dioxo-5-methyl pyrimidine. The result is ayield of 9-(2-(S) hydroxymethyl-1,3-dioxolan-4-yl)-2,4-dioxo-5-methylpyrimidine having a β:α ratio of about 2:1.

Alternatively, if the desired final product is the same compound butwith opposite stereochemistry (i.e. a 2:1 mixture of β:α stereoisomersin the D-configuration). The procedure discussed above is followed.However, when following the steps of Example 23, the sugar2-(S)-benzoyloxymethyl-4-carboxyl-1,3-dioxolane is replaced with2-(R)-benzoyloxymethyl-4-carboxyl-1,3-dioxolane.

Some modifications and variations of the present invention including butnot limited to selection of enzymes with high degree of sequencehomology and optimization of reaction conditions will be obvious to aperson of ordinary skill in the art from the foregoing detaileddescription of the invention. Such modifications and variations areintended to fall within the scope of one or more embodiments of thepresent invention as defined by the following claims.

What is claimed is:
 1. A process of stereoselectively hydrolysis comprising stereoselectively hydrolyzing an anomeric mixture of β and α anomers represented by the following formula A or formula B:

wherein R₁ is selected from the group consisting of C₁₋₆ alkyl and C₆₋₁₅ aryl, and Bz is benzoyl, with an enzyme selected from the group consisting of Protease N (Bacillus subtilis protease), Alcalase® (Subtilisin Carlsberg protease), Savinase® (Bacillus lentus subtilisin protease), ChiroCLEC-BL (Bacillus licheniformis Subtilisin protease), PS-30 (Pseudomonas cepacia lipase), and ChiroCLEC-PC (Pseudomonas cepacia lipase) to stereoselectively hydrolyze predominantly one anomer to form a product wherein R₁ is replaced with H.
 2. A process according to claim 1, further comprising separating said product from unhydrolyzed starting material to produce a second mixture.
 3. A process of claim 2, further comprising acylating the second mixture to produce an acylated second mixture.
 4. A process of claim 1, wherein the step of hydrolyzing results in the starting material having an anomeric purity of at least 97%.
 5. A process of claim 1, wherein the step of hydrolyzing results in the starting material having an anomeric purity of at least 98%.
 6. A process of claim 1, wherein the step of hydrolyzing results in the starting material having an anomeric purity of at least 98.5%.
 7. A process of claim 1, wherein the step of hydrolyzing results in the starting material having an anomeric purity of at least 98.8%.
 8. A process of claim 1, wherein the step of hydrolyzing results in the product having an anomeric purity of at least 97%.
 9. A process of claim 1, wherein the step of hydrolyzing results in the product having an anomeric purity of at least 98%.
 10. A process of claim 1, wherein the step of hydrolyzing results in the product having an anomeric purity of at least 98.5%.
 11. A process of claim 1, wherein the step of hydrolyzing results in the product having an anomeric purity of at least 98.8%.
 12. A process of claim 1, wherein R₁ is methyl.
 13. A process for stereoselectively producing a dioxolane nucleoside analogue from an anomeric mixture of β and α anomers represented by the following formula A or formula B:

wherein R₁ is methyl and Bz is benzoyl, the process comprising: stereoselectively hydrolyzing said mixture with an enzyme selected from the group consisting of Protease N (Bacillus subtilis protease), Alcalase® (Subtilisin Carlsberg protease), Savinase® (Bacillus lentus subtilisin protease), ChiroCLEC-BL (Bacillus licheniformis Subtilisin protease), PS-30 (Pseudomonas cepacia lipase), and ChiroCLEC-PC (Pseudomonas cepacia lipase) to stereoselectively hydrolyze predominantly one anomer to form a product wherein R₁ is replaced with H; separating the product from unhydrolyzed starting material to produce a second mixture; stereoselectively replacing the functional group at the C4 position with a purinyl or pyrimidinyl or derivative thereof by reacting said second mixture with a purine or pyrimidine base or derivative thereof. 